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Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors ([IP.sub.3]Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5-7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The [IP.sub.3]R inhibitors Xestospongin B or C (5 [micro]M) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10-20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three [IP.sub.3]R isoforms were present in adult muscle. [IP.sub.3]R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that [IP.sub.3]R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype. doi/ 10.1085/jgp.200910397

Heat shock proteins (HSPs) are a conserved family of cytoprotective polypeptides, synthesized by cells in response to stress. Hsp70 and heine oxygenase 1 (Hmox-1) are induced by a variety of cellular stressors in skeletal muscle, playing a role in long-term adaptations and muscle fibers regeneration. Though HSPs expression after exercise has been intensely investigated, the molecular mechanisms concerning Hsp70 and Hmox-1 induction are poorly understood. The aim of this work was to investigate the involvement of calcium in Hsp70 and Hmox-1 expression upon depolarization of skeletal muscle cells. We observed that depolarization of myotubes increased both mRNA levels and protein expression for Hsp70 and Hmox-1. Stimulation in the presence of intracellular calcium chelator BAPTA-AM resulted in a complete inhibition of Hsp70-induced expression. It is known that inositol-l,4,5-trisphophate ([IP.sub.3])-mediated slow [Ca.sup.2+] transients, evoked by membrane depolarization, are involved in the regulation of gene expression. Here we demonstrated that inhibition of [IP.sub.3]-dependent calcium signals decreased both Hsp70 mRNA induction and Hsp70 and Hmox-1 protein expression. Inhibitors of calcium-dependent protein kinase C also abolished Hsp70 mRNA induction. Our results provide evidence that membrane depolarization increases Hsp70 and Hmox-1 expression in cultured skeletal muscle cells, which the effect is critically dependent on [Ca.sup.2+] released from [IP.sub.3]-sensitive intracellular stores and that it involves PKC as an upstream effector in Hsp70 mRNA-induced expression. myotubes; excitation-transcription; inositol-l,4,5-trisphophate receptors; heat shock proteins; muscle gene expression

Depolarization of skeletal muscle cells triggers intracellular [Ca.sup.2+] signals mediated by ryanodine and inositol 1,4,5-trisphosphate ([IP.sup.3]) receptors. Previously, we have reported that [K.sup.+]-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through [IP.sub.3]-dependent [Ca.sup.2+] release, whereas NF-[kappa]B activation is elicited by both ryanodine and [IP.sub.3] receptor-mediated [Ca.sup.2+] signals. We have further shown that field stimulation with electrical pulses results in an NF-[kappa]B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both [K.sup.+] and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The [Ca.sup.2+] source for NFAT activation is through release by ryanodine receptors and extracellular [Ca.sup.2+] entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-[kappa]B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both [K.sup.+]-induced depolarization and electrical stimulation increased mRNA levels of the type 1 [IP.sub.3] receptor mediated by calcineurin activity, which suggests that depolarization may regulate [IP.sub.3] receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on [Ca.sup.2+] release and triggered by the same voltage sensor but activating different intracellular release channels. nuclear factor of activated T cells transcription; nuclear factor of activated T cells translocation; calcineurin; inositol 1,4,5-trisphosphate receptor; ryanodine receptor

Depolarization of skeletal muscle cells by either high external [K.sup.+] or repetitive extracellular field potential pulses induces calcium release from internal stores. The two components of this release are mediated by either ryanodine receptors or inositol 1,4,5-trisphosphate (I[P.sub.3]) receptors and show differences in kinetics, amplitude, and subcellular localization. We have reported that the transcriptional regulators including ERKs, cAMP/[Ca.sup.2+]-response element binding protein, c-fos, c-jun, and egr-1 are activated by [K.sup.+]-induced depolarization and that their activation requires I[P.sup.3]-dependent calcium release. We presently describe the activation of the nuclear transcription factor NF-[kappa]B in response to depolarization by either high [K.sup.+] (chronic) or electrical pulses (fluctuating). Calcium transients of relative short duration activate an NF-[kappa]B reporter gene to an intermediate level, whereas long-lasting calcium increases obtained by prolonged electrical stimulation protocols of various frequencies induce maximal activation of NF-[kappa]B. This activation is independent of extracellular calcium, whereas calcium release mediated by either ryanodine or I[P.sub.3] receptors contribute in all conditions tested. NF-[kappa]B activation is mediated by I[kappa]B[alpha] degradation and p65 translocation to the nucleus. Partial blockade by N-acetyl-L-cysteine, a general antioxidant, suggests the participation of reactive oxygen species. Calcium-dependent signaling pathways such as those linked to calcineurin and PKC also contribute to NF-[kappa]B activation by depolarization, as assessed by blockade through pharmacological agents. These results suggest that NF-[kappa]B activation in skeletal muscle cells is linked to membrane depolarization and depends on the duration of elevated intracellular calcium. It can be regulated by sequential activation of calcium release mediated by the ryanodine and by I[P.sub.3] receptors. electrical stimulation; transcription; intracellular calcium stores doi: 10.1152/ajpcell.00320.2006.

Contracting skeletal muscle produces and releases interleukin-6 (IL-6) in high amounts. Nevertheless, the mechanisms underlying IL-6 expression are not understood. Because inositol-l,4,5-trisphosphate (I[P.sub.3])- mediated slow [Ca.sup.2+] signals evoked by depolarization of skeletal myotubes appears to play a role in the regulation of gene expression, we examined its involvement on IL-6 transcription. With the use of semiquantitative RT-PCR, we have shown that [K.sup.+] depolarization of myotubes induces a transient increase in IL-6 mRNA level, which peaks at 3-4 h and is independent of extracellular [Ca.sup.2+]. Inhibitors of I[P.sub.3]-dependent [Ca.sup.2+] signals, like 2-aminoethoxydiphenyl borate (2-APB) and U-73122, decreased activation of IL-6 gene expression as did [Ca.sup.2+] signals inhibitor BAPTA-AM, whereas ryanodine, a fast [Ca.sup.2+] transient inhibitor, had no effect on IL-6 induction. Depolarization of myotubes transiently transfected with a reporter gene construct, containing 651 bp of IL-6 promoter, induced a twofold increase in promoter activity, which was abolished by either 2-APB or U-73122 and remained unaffected after ryanodine treatment. Sitedirected mutagenesis of parental construct allowed us to identify activator protein-1 and NF-[kappa]B sequences as regulatory elements involved in IL-6 upregulation. Our results provide evidence for involvement of I[P.sub.3]-mediated [Ca.sup.2+] signals on IL-6 transcription in skeletal muscle cells. myotubes; membrane potential; intracellular [Ca.sup.2]; cytokines; gene expression

The signaling mechanisms by which skeletal muscle electrical activity leads to changes in gene expression remain largely undefined. We have reported that myotube depolarization induces calcium signals in the cytosol and nucleus via inositol 1,4,5-trisphosphate ([IP.sub.3]) and phosphorylation of both ERK1/2 and cAMP-response element-binding protein (CREB). We now describe the calcium dependence of P-CREB and P-ERK induction and of the increases in mRNA of the early genes c-fos, c-jun, and egr-1. Increased phosphorylation and early gene activation were maintained in the absence of extracellular calcium, while the increase in intracellular calcium induced by caffeine could mimic the depolarization stimulus. Depolarization performed either in the presence of the [IP.sub.3] inhibitors 2-aminoethoxydiphenyl borate or xestospongin C or on cells loaded with BAPTA-AM, in which slow calcium signals were abolished, resulted in decreased activation of the early genes examined. Both early gene activation and CREB phosphorylation were inhibited by ERK phosphorylation blockade. These data suggest a role for calcium in the transcription-related events that follow membrane depolarization in muscle cells. myotubes; signal transduction; inositol 1,4,5-trisphosphate

The dihydropyridine receptor (DHPR), normally a voltage-dependent calcium channel, functions in skeletal muscle essentially as a voltage sensor, triggering intracellular calcium release for excitation-contraction coupling. In addition to this fast calcium release, via ryanodine receptor (RYR) channels, depolarization of skeletal myotubes evokes slow calcium waves, unrelated to contraction, that involve the cell nucleus (Jaimovich, E., R. Reyes, J.L. Liberona, and J.A. Powell. 2000. Am. J. Physiol. Cell Physiol. 278:C998-C1010). We tested the hypothesis that DHPR may also be the voltage sensor for these slow calcium signals. In cultures of primary rat myotubes, 10 [micro]M nifedipine (a DHPR inhibitor) completely blocked the slow calcium (fluo-3-fluorescence) transient after 47 mM [K.sup.+] depolarization and only partially reduced the fast [Ca.sup.2+] signal. Dysgenic myotubes from the GLT cell line, which do not express the [[alpha].sub.1] subunit of the DHPR, did not show either type of calcium transient following depolarization. After transfection of the [[alpha].sub.1] DNA into the GLT cells, [K.sup.+] depolarization induced slow calcium transients that were similar to those present in normal [C.sub.2][C.sub.12] and normal NLT cell lines. Slow calcium transients in transfected cells were blocked by nifedipine as well as by the G protein inhibitor, pertussis toxin, but not by ryanodine, the RYR inhibitor. Since slow [Ca.sup.2+] transients appear to be mediated by I[P.sub.3], we measured the increase of I[P.sub.3] mass after [K.sup.+] depolarization. The I[P.sub.3] transient seen in control cells was inhibited by nifedipine and was absent in nontransfected dysgenic cells, but [[alpha].sub.1]-transfected cells recovered the depolarization-induced I[P.sub.3] transient. In normal myotubes, 10 [micro]M nifedipine, but not ryanodine, inhibited c-jun and c-fos mRNA increase after [K.sup.+] depolarization. These results suggest a role for DHPR-mediated calcium signals in regulation of early gene expression. A model of excitation-transcription coupling is presented in which both G proteins and I[P.sub.3] appear as important downstream mediators after sensing of depolarization by DHPR. KEY WORDS: dysgenic cells * gene expression * calcium waves * inositol trisphosphate * excitation transcription